This invention relates to iron-binding polymers, particularly polymers administered orally to decrease the absorption of dietary iron from the gastrointestinal tract.
Reduced uptake of dietary iron is clinically important in several related metabolic disorders. In patients with hemochromatosis too much dietary iron is absorbed and patients experience iron overload. Genetic hemochromatosis is due to a scmatic gene mutation. While tissue damage is greatest in individuals who are homozygous for the defective gene, reduction of iron uptake is also desirable in patients who are heterozygous for the implicated mutation (Finch et al., N. Engl. J. Med., 306:1520 (1982)). Acquired hemochromatosis includes conditions characterized by tissue injury associated with iron overload, where disease processes other than genetic mutations cause the exacerbated iron uptake. Examples of such disease include iron-loading anemias, such as thalassemia and sideroblastic anemia, as well as certain types of liver dysfunction (Finch et al., N. Engl J. Med., 306:1520 (1982)). The massive deposits of iron in body tissues cause similar organ failure in both genetic and acquired hemochromatosis.
Until recently, relatively high iron levels were considered desirable in all individuals. However, increased rates of heart disease are now known to be associated with elevated serum ferritin levels (an indicator of the body burden of iron). In the heterozygous state of hemochromatosis, for example, the degree of iron overload is not sufficient to lead to the traditional symptoms of overload, including abdominal pain, hepatomegaly, diabetes, impotence, and gray pigmentation of the skin. The iron overload may be sufficient, however, to lead to increased probability of heart disease such as congestive heart failure.
A typical adult man has 4-6 g of iron in his body, and absorbs approximately 1 mg of the 10-20 mg of iron available from his daily diet. Iron is absorbed in two basic forms, free iron and heme-bound iron. Free iron can be in either the Ferrous (FExe2x88x922) or ferric (Fexe2x88x923) forms, and can be complexed to various organic and inorganic dietary ingredients (such as phosphate, phytase and citrate). The two forms of free iron are absorbed equally well provided that they both remain in an ionized form, and not in the easily formed and insoluble hydroxides. A typical adult diet contains approximately 1.6 mg of heme-bound iron and 13 mg of free iron. Heme-bound iron, while present in smaller amounts in the diet than free iron, is more readily absorbed than free iron. Approximately 23% of heme-bound iron is available for absorption, while the absorbable fraction of dietary free iron ranges from 3-8%, depending on the other constituents of the diet. The result of these factors is that both heme-bound and free iron contribute significantly to dietary iron uptake.
Iron is absorbed primarily in the proximal segments of the small intestine. It is absorbed by the mucosal cells, processed into appropriate forms, and released into the plasma.
In general, the invention features a method of reducing dietary iron absorption in a patient which involves oral administration of a therapeutically effective amount of one or more iron-binding polymers that are non-toxic and stable once ingested.
By xe2x80x9cnon-toxicxe2x80x9d it is meant that when ingested in therapeutically effective amounts neither the polymers nor any ions released into the body upon ion exchange are harmful.
By xe2x80x9cstablexe2x80x9d it is meant that when ingested in therapeutically effective amounts the polymers do not dissolve or otherwise decompose to form potentially harmful by-products, and remain substantially intact so that they can transport bound iron out of the body.
By xe2x80x9csaltxe2x80x9d it is meant that the nitrogen group in the repeat unit is protonated to create a positively charged nitrogen atom associated with a negatively charged counterion.
By xe2x80x9calkylating agentxe2x80x9d it is meant a reactant which, when reacted with polymer, causes an alkyl group or derivative thereof (e.g., a substituted alkyl, such as an aralkyl, hydroxyalkyl, alkylammonium salt, alkylamide, or combination thereof) to be covalently bound to one or more of the nitrogen atoms of the polymer.
In one preferred embodiment the polymer includes primary, secondary, tertiary, or quaternary amines. These amines may include xe2x80x94NR3+, where each R group, independently, is H or a lower alkyl or aryl group.
One example of a preferred polymer is characterized by a repeating unit having the formula 
or a copolymer thereof, wherein n is an integer and each R, independently, is H or a substituted or unsubstituted alkyl, alkylamino, or aryl.
A second example of a preferred the polymer is characterized by a repeating unit having the formula 
or a copolymer thereof, wherein n is an integer, each R, independently, is H or a substituted or unsubstituted alkyl, alkylamino, or aryl group, and each Xxe2x88x92 is an exchangeable negatively charged counterion.
A third example of a preferred polymer is a copolymer characterized by a first repeating unit having the formula 
wherein n is an integer, each R, independently, is H or a substituted or unsubstituted alkyl, alkylamino, or aryl group and each X is an exchangeable negatively charged counterion; and further characterized by a second repeating unit having the formula 
wherein each n, independently, is an integer and each R, independently, is H or a substituted or unsubstituted alkyl, alkylamino, or aryl group.
A fourth example of a preferred polymer is characterized by a repeating unit having the formula 
or a copolymer thereof, wherein n is an integer, and R is H or a substituted or unsubstituted alkyl, alkylamino, or aryl group.
One example of a copolymer according to the fourth aspect of the invention is characterized by a first repeating unit having the formula 
wherein n is an integer, and R is H or a substituted or unsubstituted alkyl, alkylamino or aryl group; and further characterized by a second repeating unit having the formula 
wherein each n, independently, is an integer and R is H or a substituted or unsubstituted alkyl, alkylamino, or aryl group.
A fifth example of a polymer is characterized by a repeating group having the formula 
or a copolymer thereof, wherein n is an integer, and each R1 and R2, independently, is H or a substituted or unsubstituted alkyl, alkylamino, or aryl and each Xxe2x88x92 is an exchangeable negatively charged counterion.
As an example of another preferred polymer, according to the fifth aspect of the invention, at least one of the R groups is a hydrogen atom.
A sixth example of a preferred polymer is characterized by a repeat unit having the formula 
or a copolymer thereof, where n is an integer, each R1 and R2, independently, is H, a substituted or unsubstituted alkyl, alkylamino, or aryl group.
A seventh example of a preferred polymer is characterized by a repeat unit having the formula 
or a copolymer thereof, wherein n is an integer, each R1, R2 and R3, independently, is H, a substituted or unsubstituted alkyl, alkylamino, aryl group and each Xxe2x88x92 is an exchangeable negatively charged counterion.
An eighth example of a preferred polymer is characterized by one or more crosslinked polymers comprising
(1) a hydrophobic co-monomer and
(2) a repeat unit having the formula 
or copolymer thereof, where n is an integer; R1 is H or a C1-C20 alkyl group; 
Z is O, NR3, S, or (CH2)m; m=0-10; R3 is H or a C1-C20 alkyl group; and R2 is 
where p=0-10, and each R4, R5, and R6, independently, is H, a C1-C20 alkyl group, or an aryl group.
A ninth example of a preferred polymer is characterized by the reaction product of:
(a) one or more crosslinked polymers comprising a repeat unit having the formula: 
or copolymer thereof, where n is an integer; R1 is H or a C1-C20 alkyl group; 
Z is O, NR3, S, or (CH2)m; m=0-10; R3 is H or a C1-C20 alkyl group; and R2 is 
where p=0-10, and each R4, R5, and R6, independently, is H, a C1-C20 alkyl group or an aryl group alkylated reaction products or copolymers thereof.
A tenth example of a preferred polymer is characterized by the reaction product of:
a) one or more crosslinked polymers characterized by a repeat unit selected from the group consisting of: 
and salts and copolymers thereof, where n is an integer and each R, independently, is H or a C1-C20 alkyl group; and
b) at least one alkylating agent.
An eleventh example of a preferred polymer is characterized by an amine polymer, comprising:
a) a first substituent, bound to an amine of the amine polymer, that includes a hydrophobic moiety; and
b) a second substituent, bound to an amine of the amine polymer, that includes a quaternary amine-containing moiety.
A twelfth example of a preferred polymer is characterized by an amine polymer, comprising a substituent bound to an amine of the amine polymer, the substituent including a quaternary amine-containing moiety having at least one hydrophobic substituent.
The polymers of the invention may be crosslinked.
In another aspect, the invention features a therapeutic composition suitable for oral administration, including a therapeutically effective amount of at least one polymer that binds dietary iron, where the polymer is non-toxic and stable once ingested. By xe2x80x9ctherapeutically effectivexe2x80x9d is meant a composition which, when administered to a patient causes decreased absorption of dietary iron.
The invention provides an effective treatment for decreasing the absorption of dietary iron, and thereby reducing the patient""s total body iron stores. The compositions are non-toxic and stable when ingested in therapeutically effective amounts.
Other features and advantages will be apparent from the following description of the preferred embodiments and from the claims.
The polymers employed in the method described herein have been described by Applicants in copending application Ser. Nos. 08/065,546, filed May 20, 1993, 08/258,431, filed Jun. 10, 1994, 08/460,980, filed Jun. 5, 1995, 08/461,298, filed Jun. 5, 1995, 08/469,659, filed Jun. 6, 1995, 08/471,747, filed Jun. 6, 1995, 08/471,769, filed Jun. 6, 1995 and 08/482,969, filed Jun. 7, 1995 the contents of which are incorporated herein by reference in their entirety.
The polymers of the invention generally include hydrophilic anion exchange resins, particularly aliphatic amine polymers. The xe2x80x9caminexe2x80x9d group can be present in the form of a primary, secondary or tertiary amine, quaternary ammonium salt, amidine, guanadine, hydrazine, or combinations thereof. The amine can be within the linear structure of the polymer (such as in polyethylenimine or a a condensation polymer of a polyaminoalkane, e.g. diethylenetriamine, and a crosslinking agent, such as epichlorohydrin) or as a functional group pendant from the polymer backbone (such as in polyallylamine, polyvinylamine or poly)aminoethyl)acrylate).
In one aspect, the polymer is characterized by a repeating unit having the formula 
or a copolymer thereof, wherein n is an integer and each R, independently, is H or a substituted or unsubstituted alkyl, such as a lower alkyl (e.g., having between 1 and about 20 carbon atoms), alkylamino (e.g., having between 1 and about 20 carbon atoms, such as ethylamino) or aryl (e.g., phenyl) group.
In another aspect, the polymer is characterized by a repeating unit having the formula 
or a copolymer thereof, wherein n is an integer, each R, independently, is H or a substituted or unsubstituted alkyl, (e.g., having between 1 and about 20 carbon atoms), alkylamino (e.g., having between 1 and about 20 carbon atoms, such as ethylamino) or aryl (e.g., phenyl) group, and each Xxe2x88x92 is an exchangeable negatively charged counterion.
One example of a copolymer of the invention is characterized by a first repeating unit have the formula 
wherein n is an integer, each R, independently, is H or a substituted or unsubstituted alkyl (e.g., having between 1 and about 20 carbon atoms), alkylamino (e.g., having between 1 and about 20 carbon atoms, such as ethylamino) or aryl group (e.g., phenyl), and each Xxe2x88x92 is an exchangeable negatively charged counterion; and further characterized by a second repeating unit having the formula 
wherein each n, independently, is an integer and each R, independently, is H or a substituted or unsubstituted alkyl (e.g., having between 1 and about 20 carbon atoms), alkylamino (e.g., having between 1 and about 20 carbon atoms, such as ethylamino) or aryl group (e.g., phenyl).
In yet another aspect, the polymer characterized by a repeating unit having the formula 
or a copolymer thereof, wherein n is an integer, and R is H or a substituted or unsubstituted alkyl (e.g., having between 1 and about 20 carbon atoms), alkylamino (e.g., having between 1 and about 20 carbon atoms, such as ethylamino) or aryl group (e.g., phenyl).
Another example of a copolymer of the invention is characterized by a first repeating unit having the formula 
wherein n is an integer, and R is H or a substituted or unsubstituted alkyl (e.g., having between 1 and about 20 carbon atoms), alkylamino (e.g., having between 1 and about 20 carbon atoms such as ethylamino) or aryl group (e.g., phenyl); and further characterized by a second repeating unit having the formula 
wherein each n, independently, is an integer and R is H or a substituted or unsubstituted alkyl (e.g., having between 1 and about 20 carbon toms), alkylamino (e.g., having between 1 and about 20 carbon atoms, such as ethylamino) or aryl group (e.g., phenyl).
In still another aspect, the polymer is characterized by a repeating group having the formula 
or a copolymer thereof, wherein n is an integer, and each R1 and R2, independently, is H or a substituted or unsubstituted alkyl (e.g., having between 1 and about 20 carbon atoms), and alkylamino (e.g., having between 1 and about 20 carbon atoms, such as ethylamino) or aryl group (e.g., phenyl), and each Xxe2x88x92 is an exchangeable negatively charged counterion. A preferred polymer has at least one hydrogen as one of the R groups.
In still another aspect, the polymer is characterized by a repeat unit having the formula 
or a copolymer thereof, where n is an integer, each R1 and R2, independently, is H, a substituted or unsubstituted alkyl group containing 1 to about 20 carbon atoms, an alkylamino group (e.g., having between 1 and about 20 carbon atoms, inclusive, such as ethylamino), or an aryl group containing 6 to 20 atoms (e.g., phenyl).
In yet another aspect, the polymer is characterized by a repeat unit having the formula 
or a copolymer thereof, wherein n is an integer, each R1, R2 and R3, independently, is H, a substituted or unsubstituted alkyl group containing 1 to 20 carbon atoms, an alkylamino group (e.g., having between 1 and about 20 carbon atoms, such as ethylamino), or an aryl group containing 6 to 20 atoms (e.g., phenyl), and each Xxe2x88x92 is an exchangeable negatively charged counterion.
In each case, the R groups can carry one or more substituents. Suitable substituents include therapeutic anionic groups, e.g., quaternary ammonium groups, or amine groups, e.g., primary and secondary alkyl or aryl amines. Examples of other suitable substituents include hydroxy, onercapto, alkoxy, carboxamide, sulfonamide, halogen, alkyl, aryl, oxime, hydrazine, guanadine, urea, and carboxylic acid esters, for example.
The present invention also includes reaction products characterized by repeat unit having the formula: 
or copolymer thereof, where n is an integer; R1 is H or an alkyl group (which may be straight chain or branched, substituted or unsubstituted, e.g., a C1-C20 alkyl, such as methyl); 
Z is O, NR3, S, or (CH2)m; m=0-10; R3 is H or an alkyl group (which may be straight chain or branched, substituted or unsubstituted, e.g., C1-C20 alkyl, such as methyl); and R2 is 
where p=0-10, and each R4, R5, and R6, independently, is H, an alkyl group (which may be straight chain or branched, substituted or unsubstituted, e.g., C1-C20 such as methyl), or an aryl group (e.g., having one or more rings and which may be substituted or unsubstituted, e.g., phenyl, naphthyl, imidazolyl, or pryridyl).
Polymers of the invention also include those polymers characterized by the formula: 
where R1 is hydrogen or methyl, Z1 is O or NR3, R3 is hydrogen or an alkyl group, R4, R5 and R6 are, independently, hydrogen or methyl, and p=2-10.
In a preferred embodiment, the polymer is characterized by the formula: 
where R1 is hydrogen or methyl, R4, R5 and R6 are, independently hydrogen or alkyl and p=0-2.
The polymer can also be characterized by the formula 
wherein R1 is hydrogen or methyl, R3 is hydrogen or an alkyl group, R4, R5 and R6 are, independently, hydrogen or methyl, and p=2-10.
The polymers also include heteropolymers of two or more of the above.
The polymer further can include one or more hydrophilic or hydrophobic co-monomers, e.g., styrene, vinyl naphthalene, ethyl vinylbenzene, N-alkyl and N-aryl derivatives of acrylamide and methacrylamide, alkyl and aryl acrylates, alkyl and aryl methacrylates, 4-vinybiphenyl, 4-vinyl-anisole, 4-aminostyrene, and fluorinated derivatives of any of these co-monomers (e.g., p-fluorostyrene, pentafluoro-styrene, hexafluoroisopropylacrylate, hexafluorobutyl-methacrylate, or heptadecafluoro-decylmethacrylate). For example, the co-monomer can be an alkylated derivative or other derivative of one or more of the above mentioned formulae. The alkyl groups are preferably C1-C20 (e.g., C1-C20 alkyl groups, and may be straight chain, branched, or cyclic (e.g., cyclohexyl), and may further be substituted or unsubstituted. The aryl groups preferably have one or more rings and may be substituted or unsubstituted, e.g., phenyl, naphthyl, imidazolyl, or pryridyl. The polymer may also include one or more positively charged or amine co-monomers, e.g., vinyl pyridine, dimethylaminomethyl styrene, or vinyl imidazole.
Another example of a preferred polymer is characterized by a repeat unit having the formula 
or copolymer thereof. The polymer may further include, as a co-monomer, ethyl vinylbenzene.
In yet another example of a preferred polymer is characterized by a repeat unit having the formula 
or copolymer thereof.
In still yet another example of a preferred polymer is characterized by a repeat unit having the formula 
or copolymer thereof. The polymer may also include, as a co-monomer, styrene or a fluorinated derivative thereof.
In another aspect, the invention features polymers and a method for removing iron from a patient by ion exchange that includes administering to the patient a therapeutically effective amount of one or more crosslinked polymers characterized by a repeat unit having the formula 
or copolymer thereof, where n is an integer; R1 is H or a C1-C20 alkyl group; L is xe2x80x94NHxe2x80x94 or 
G is 
and each R2, R3, and R4, independently, is H, A C1-C20 alkyl group, or an aryl group. The polymers are preferably non-toxic and stable once ingested.
One example of a preferred polymer is characterized by a repeat unit having the formula 
or copolymer thereof. The polymer may further include, as a co-monomer, styrene or a fluorinated derivative thereof.
Another example of a preferred polymer is characterized by a repeat unit having the formula 
or copolymer thereof.
Optionally, the polymer includes one or more co-monomers that increase the overall hydrophobicity of the polymer. Because iron-heme complexes are hydrophobic, the hydrophobic co-monomer can aid in maximizing the selectivity of the interaction of the polymer with the heme molecule.
Examples of suitable hydrophobic co-monomers include, e.g., acrylamide, methacrylamide, and N-alkyl (e.g., methyl, ethyl, isopropyl, butyl, hexyl, dodecyl, cyclohexyl, dicyclohexyl) and N-aryl (e.g., phenyl diphenyl) derivatives thereof; alkyl and aryl acrylates and methacrylates (e.g., ethyl, propyl, butyl, dodecyl), and fluorinated derivatives thereof (e.g., hexafluoroisopropyl acrylate, hexafluorobutyl methacrylate, heptadecafluorodecyl acrylate); styrene and derivatives thereof (e.g., dimethylaminomethyl styrene, 4-aminostyrene, and fluorinated derivatives, e.g., p-fluorostyrene, pentafluorosstyrene); ethylvinylbenzene; vinyl naphthalene; vinyl pyridine; vinyl imidazole; 4-vinylbiphenyl; 4,4-vinylanisole; and combinations thereof. The amount of co-monomer used in the preparation of these polymers is from 0 to 75% by weight.
The level of hydrophobicity desired can also be achieved simply by appropriate choice of crosslinking co-monomer. For example, divinylbenzene is a suitable crosslinking co-monomer and is hydrophobic as well. In addition, the main xe2x80x9cimpurityxe2x80x9d in divinylbenzene is ethylvinylbenzene, a hydrophobic, polymerizable monomer which will also contribute to the overall hydrophobicity of the polymer. Other hydrophobic crosslinking co-monomers include bisphenol A diacrylate and bisphenol A dimethacrylate.
An additional example of a preferred polymer includes the products of one or more crosslinked polymers having the formulae:
(a) one or more crosslinked polymers characterized by a repeat unit selected from the group consisting of: 
xe2x80x83and salts and copolymers thereof, where n is an integer and each R, independently, is H or a substituted or unsubstituted alkyl group (e.g., C1-C20 alkyl); and
(b) at least one alkylating agent. The reaction product is preferably non-toxic and stable once ingested. The polymers are, in one embodiment, crosslinked. The level of crosslinking makes the polymers completely insoluble and thus limits the activity of the alkylated reaction product to the gastrointestinal tract only. Thus, the compositions are non-systemic in the activity and will lead to reduced side-effects in the patient.
An example of preferred polymer is characterized by a repeat unit having the formula 
or a salt or copolymer thereof; wherein x is zero or an integer between about 1 to 4.
Another example of a preferred polymer is characterized by a repeat unit having the formula
(NHxe2x80x94CH2CH3)n 
or a salt or copolymer thereof.
Still yet another example of a preferred polymer is characterized by a repeat unit having the formula
(NHxe2x80x94CH2CH2xe2x80x94NHxe2x80x94CH2CH3xe2x80x94NHxe2x80x94CH2CHOHxe2x80x94CH2)n 
or a salt or copolymer thereof.
The amine polymers of the invention can include distinct first and second substituents. The first substituent is bound to an amine of the amine polymer and can include a hydrophobic moiety. The second substituent is bound to an amine of the amine polymer and includes a quaternary amine-containing moiety. It is to be understood that the first and second substituents can be bound to the same amine and/or different amines of the amine polymer. The amine polymers of the invention are particularly suitable for binding iron in mammals by oral administration of the polymer. A particularly suitable form for oral administration of the amine polymer is that which will form a gel in the stomach of a patient.
Suitable methods by which the amine polymer of the invention can be formed include polymerization of an amine monomer to form a homopolymer. Examples of this method include polymerization of allylamine, ethyleneimine, vinylamine, 1,2-diaminoethene, aminoethylacrylamide, aminopropylacrylate, or p-aminomethylstyrene, to form their respective homopolymers.
Another method involves copolymerizing an amine monomer with one or more additional monomers. These additional monomers include amine monomers, such as those listed above, and non-amine monomers, such as acrylamide, styrene, divinylbenzene, vinyl alcohol, or vinyl chloride. Examples include copoly(allylamine/acrylamide), copoly(vinylamine/allylamine), copoly(aminoethylacrylamide/acrylamide), and copoly(allylamine/divinylbenzene).
Still another method involves polymerization of a non-amine monomer to form a homopolymer that is subsequently chemically modified to form an amine polymer. Examples of this method include polymerization of vinyl formamide, vinyl acetamide, vinyl chloride, vinyl bromide, allyl chloride, allyl bromide, acrylamide, or acrylonitrile, to form their respective homopolymers. Each homopolymer would then be chemically altered to form an amine polymer using such reactions as hydrolysis, nucleophilic substitution, or reduction. The first four homopolymers listed above would then become poly(vinylamine) and the last four would become poly(allylamine). It is to be understood that not all of the initial non-amine monomer need be chemically altered, resulting in an amine polymer that contains some of the initial non-amine monomers in a non-amine state.
Another method involves copolymerizing a non-amine monomer with one or more additional monomers. These additional monomers could include amine monomers and non-amine monomers. The resulting copolymer would then be chemically altered. Examples would include copolymerization of acrylamide and styrene, followed by reduction to form copoly(allylamine/styrene), copolymerization of acrylonitrile and vinyl formamide, followed by reduction and hydrolysis, to form copoly(allylamine/vinylamine), and copolymerization of acrylonitrile and allylamine, followed by reduction, to form poly(allylamine). It is to be understood that not all of the initial non-amine monomer will be therefore chemically altered, resulting in an amine polymer that contains some of the initial non-amine monomers in a non-amine state.
Still another method involves forming an amine polymer through a condensation mechanism. Examples of this method would include reaction of diethylenetriamine and epichlorohydrin, 1,3-dibromopropane and ethylenediamine, spermine and 1,4-butanediol diglycidyl ether, or tris(2-aminoethyl)amine and 1,10-dibromodecane.
Each of these amine polymers typically has a molecular weight greater than 2,000. Examples of resulting suitable amine polymers include poly(vinylamine), poly(allylamine), and poly(ethyleneimine). A preferred amine polymer is poly(allylamine).
The polymers are preferably crosslinked, in some cases by adding a crosslinking co-monomer to the reaction mixture during polymerization. Examples of suitable crosslinking co-monomers are diacrylates and dimethacrylates (e.g., ethylene glycol diacrylate, propylene glycol diacrylate, butylene glycol diacrylate, ethylene glycol dimethacrylate, propylene glycol dimethacrylate, butylene glycol dimethacrylate, polyethyleneglycol dimethacrylate, polyethyleneglycol diacrylate, methylene bisacrylamide, methylene bismethacrylamide, ethylene bisacrylamide, ethylenebismethacrylamide, ethylidene bisacrylamide, divinyl benzene, biphenol A dimethacrylate, and bisphenol A diacrylate. These crosslinking co-monomers are either commercially available or are prepared as described in Mandeville et al., xe2x80x9cProcess for Adjusting Ion Concentration in a Patient and Compositions Therefor,xe2x80x9d U.S. Ser. No. 08/065,113, filed May 20, 1993, assigned to the same assignee as the present application and hereby incorporated by reference.
The amount of crosslinking co-monomer is typically between 1.0 and 25 weight %, based upon combined weight of crosslinking agent and monomer.
In some cases the polymers are crosslinked after polymerization. One method of obtaining such crosslinking involves reaction of the polymer with difunctional crosslinkers, such as epichlorohydrin, succinyl dichloride, the diglycidal ether or bisphenol A, pyromellitic dianhydride, toluene diisocyanate, and ethylenediamine. A typical example is the reaction of poly(ethyleneimine) with epichlorohydrin. In this example the epichlorohydrin (1-100 parts) is added to a solution containing polyethyleneimine (100 parts) and heated to promote reaction. Other methods of inducing crosslinking on already polymerized materials includes, but is not limited to, exposure to ionizing radiation, ultraviolet radiation, electron beams, radicals, and pyrolysis.
Crosslinking of the polymer can be achieved by reacting the polymer with a suitable crosslinking agent in an aqueous caustic solution at about 25xc2x0 C. for a period of time of about eighteen hours to thereby form a gel. The gel is then combined with water or dried to form a particulate solid. The particulate solid can then be washed with water and dried under suitable conditions, such as a temperature of about 50xc2x0 C. for a period of time of about eighteen hours.
The amine polymer can be alkylated. One or more alkylating agents can be employed to react with the amine polymer to form substituents on the amine polymer. In one example the first substituent is bound to an amine of the amine polymer, and includes a hydrophobic moiety. Examples of suitable hydrophobic moieties are those which include alkyl groups of at least six carbons. In one embodiment, the hydrophobic moiety includes an alkyl group of between about eight and twelve carbons. Specific examples of suitable hydrophobic moieties include alkyl halides, such as n-hexyl halide, n-octyl halide, n-decyl halide, n-dodecyl halide, n-tetradecyl halide, n-octadecyl halide, and combinations thereof. Other examples include: a dihaloalkane that includes an alkyl group of at least six carbons (e.g., a 1,10-dihalodecane); an hydroxyalkyl halide (e.g., an 11-halo-1-undecanol); an aralkyl halide (e.g., a benzyl halide); etc. The alkylating agent can include a suitable leaving group, such as a halide, epoxy, tosylate, or mesylate group. In the case of, e.g., epoxy groups, the alkylation reaction causes opening of the three-membered epoxy ring.
A preferred halogen component of the alkyl halides is bromine. An example of an alkylating agent which, when reacted with the amine polymer, will cause formation of an amine polymer reaction product that includes a first substituent, as 1-bromodecane.
The amine polymer can also be alkylated with a second alkylating agent. The second alkylating agent, when reacted with the amine polymer, will result in an amine polymer reaction product that includes a second substituent that is bound to an amine of the amine polymer. The second substituent can include a quaternary amine-containing moiety. In one embodiment, the quaternary amine-containing moiety of the second substituent includes an alkyl trimethylammonium, wherein the alkyl component includes between about two and twelve carbons. Examples of preferred alkyl groups of the alkyl trimethylammonium are hexyl, octyl, and decyl groups. Examples of suitable second alkylating agents include alkyl halide trimethylammonium salts, such as (4-halobutyl) trimethylammonium salt, (6-halohexyl)trimethylammonium salt, (8-halooctyl)trimethylammonium salt, (10-halodecyl) trimethylammonium salt, (12-halododecyl)trimethylammonium salt, and combinations thereof. A particularly preferred second alkylating agent is (6-bromohexyl)trimethylammonium bromide.
The amine polymer is typically alkylated by combining the polymer with the alkylating agent is a solvent such as an organic solvent or water. Examples of suitable organic solvents include methanol, ethanol, acetonitrile, etc. A preferred organic solvent is methanol.
In another embodiment, the reaction mixture is heated over a period of about forty minutes to a temperature of about 65xc2x0 C., with stirring. Typically, an aqueous sodium hydroxide solution is intermittently added during the reaction period. Preferably, the reaction period at 65xc2x0 C. is about eighteen hours, followed by gradual cooling to a room temperature of about 25xc2x0 C. over a period of about four hours. The resulting reaction product is then filtered, resuspended in methanol, filtered again, and then washed with a suitable aqueous solution, such as two molar sodium chloride, and then with deionized water. The resultant solid product is then dried under suitable conditions, such as at a temperature of about 60xc2x0 C. in a forced-air oven. The dried solid can then be subsequently processed. Preferably, the solid is ground and passed through an 80 mesh sieve.
In one embodiment of the invention, the amine polymer is a crosslinked poly(allylamine), wherein the substituent includes (3-bromopropyl)dodecyldimethylammonium bromide. Further, the particularly preferred crosslinked poly(allylamine) is crosslinked by epichlorohydrin that is present in a range of between about two and six percent of the amines of the polymer.
In another embodiment, (6-bromohexyl) trimethylammonium bromide can be formed by adding to a 5 L, three-neck flask, equipped with a mechanical stirrer, thermometer, and a condenser at xe2x88x925xc2x0 C., tetrahydrofuran (3.0 L) and 1,6-dibromohexane (1.0 kg). To this mixture is added trimethylamine (gas; 241.5 grams) over a 1 hour period. At the end of this addition the temperature is xe2x88x9240xc2x0 C. The mixture is stirred and temperature maintained at 40xc2x0 C. for 24 hours. The solid is then filtered off and rinsed with tetrahydrofuran (2.0 L). The solid is dried in a vacuum oven to yield 1070.2 grams of white solid. This solid is then used as an alkylating agent.
In another embodiment of the invention, the amine polymer is a crosslinked poly(allylamine), wherein the first substituent includes a hydrophobic alkyl, such as decyl moiety, and the second amine substituent includes an ammonium substituted alkyl such as hexyltrimethylammonium. Further, the preferred crosslinked poly(allylamine) is crosslinked by epichlorohydrin that is present in a range of between about two and six percent of the amines of the polymer.
The negatively charged counterions, Xxe2x88x92, can be organic ions, inorganic ions, or a combination thereof. The inorganic ions suitable for use in this invention include halide (especially chloride), carbonate, bicarbonate, sulfate, bisulfate, hydroxide, nitrate, persulfate and sulfite. Suitable organic ions include acetate, ascorbate, benzoate, citrate, dihydrogen citrate, hydrogen citrate, oxalate, succinate, tartrate, taurocholate, glycocholate, cholate, lactate, propionate, butyrate, ascorbate, maleate, folate, an amino acid derivative, a nucleotide, a lipid, or a phospholipid. The counterions may be the same as, or different from, each other. For example, the polymer may contain two different types of counterions, both of which are exchanged for the iron being removed. More than one polymer, each having different counterions associated with the fixed charges, may be administered as well.
In a preferred embodiment, the counterion does not have a detrimental side effect to the patient but rather is selected to have a therapeutic or nutritional benefit to the patient.
Preferably, the ions related into the body are actually beneficial to the patient. Such is the case when, for example, the exchangeable ions are natural nutrients such as amino acids, or possess a therapeutic value.
The amine polymers of the invention can be subsequently treated or combined with other materials to form compositions for oral administration of amine polymers.
The present pharmaceutical compositions are generally prepared by known procedures using well known and readily available ingredients. In making the compositions of the present invention, the amine polymer can be present alone, can be admixed with a carrier, diluted by a carrier, or enclosed within a carrier which may be in the form of a capsule, sachet, paper or other container. When the carrier serves as a diluent, it can be a solid, semi-solid or liquid material which acts as a vehicle, excipient or medium for the polymer. Thus, the composition can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, syrups, aerosols, (as a solid or in a liquid medium), soft or hard gelatin capsules, sterile packaged powders, and the like. Examples of suitable carrier, excipients, and diluents include foods, drinks, lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, methyl cellulose, methylhydroxyenzoates, propylhydroxybenzoates, propylhydroxybenzoates, and talc.
The method of the invention includes administering to a mammal, such as by oral administration, a therapeutic amount of the amine polymer having a first substituent, bound to an amine of the amine polymer, that includes a hydrophobic moiety, and a second substituent, bound to an amine of the amine polymer, that includes a quaternary amine-containing moiety. Generally, a therapeutic amount of the amine polymer is an amount of the amine polymer in a range of between about 0.1 grams/day and about 10 grams/day.
Polymers for binding free iron and heme-bound iron may be different, and their efficacies can be assessed by different tests. For these reasons the two types of iron are discussed separately.
Heme-Bound Iron
One method of sequestering heme-bound iron would involve binding it to a polymer, rendering it unable to enter the mucosal cells. The structure of heme-bound iron is shown below. 
There are several logical ways to attach this molecule to a polymer, as described below.
1. Since in the small intestine the pH would typically be around 7, the two carboxylic acid groups will likely be ionized to form negatively charged RCO2xe2x88x92 groups. If the polymer contained positively charged groups the heme could be bound by its negatively charged groups through an ion-exchange mechanism. Examples of positively charged groups (at pH 7) would include primary, secondary, tertiary, and quaternary amines.
2. The iron atom itself is also available for binding, even though four of its six sites are taken up by the heme. In natural proteins, such as hemoglobin and cytochrome C, these items are bound to by such ligands as the nitrogen group of histidine and the sulfur group of methionine. The polymer would thus incorporate one or more appropriate ligands to bind directly to the iron atom.
3. A polymer with a site that provided appropriate solvation for the various parts of the heme-iron would also effectively bind it. The heme unit incorporates a variety of organic functional groups that vary in their solvation requirements, from the various carboxylic acid groups which would be best solvated by polar, hydrogen bonding moieties to the allyl groups which would be better solvated by nonpolar, nonhydrogen-bonding moieties.
4. A preferred embodiment would include a polymer which combined two or more of these mechanisms in a single site or, alternatively, at separate sites.
In order to assess the potential of each candidate polymer a test was devised to quantitate the binding of the iron-heme unit to the polymer. This test involved stirring the polymer in a solution designed to mimic hystiologic conditions. The amount of heme chosen corresponds to 10 mg of iron (a typical daily intake) and is dissolved in 1 L of fluid (the amount typically passing out of the small intestine in one day).
A specified amount of polymer was stirred in 100 mL of this solution for three hours. The pH was adjusted to 7.0 at both the start and end of this period. The solid was then filtered off and the amount of heme still present in the solution was determined spectroscopically. For any given polymer the amount of heme remaining in the solution is a function of the amount of polymer used in the test.
As shown in the following table, the amount of one preferred polymer, poly(ammoniumbutylacrylamide) (ABA), positively correlates with the percent heme remaining after filtration.
The daily dose column is an estimate of the dose required by a person who consumes 10 mg/day of heme iron. Thus to sequester 99% of the heme iron from this individual""s diet he would have to take 1.3 g of polymer over the course of the day.
This test is extremely sensitive to the pH of the test solution, and care must be made to ensure that the pH is 7.0. As the pH is raised above pH 7, the binding drops off significantly. Further, at pH values below 7 (especially below 5.5) the heme is insoluble and precipitates. Thus the tests must be run carefully at pH 7.
In order to assess the relative binding ability of a variety of polymers, a few selected points were tested. The table below shows the data for a number of such polymers.
In order to combine the effects of ion exchange (binding method 1) with those of hydrophobicity (method 3 a series of copolymers was formed. In the first case a copolymer involving ammoniumethylacrylamide (AEA) and allylacrylamide (AA) was made with allylacrylamide portions ranging from 0% to 75%. As can be seen in the data below, the higher the proportion of allylacrylamide in the polymer the poorer the binding is. In this case the added hydrophobicity did not increase the binding.
Other polymers were also made to test the effects of hydrophobicity on binding. One set includes a comparison of an acrylamide polymer to the more hydrophobic methacrylamide equivalent. A second comparison from this set involves substitution of more hydrophobic ethyl groups for methyl groups. From these comparisons there is no clear trend concerning the effect of hydrophobicity on iron binding effectiveness.
Other comparison involving hydrophobicity come from the following list of polymers:
From these comparisons it is again shown that increased hydrophobicity does not improve iron binding. In order to make many of these comparisons some of the iron binding monomer was diluted with a nonpolar monomer. This dilution necessarily lowers the concentration of the primary monomer. Alternatively one can dilute the primary monomer with a hydrophilic monomer, thereby separating the effects of dilution from those of increased hydrophobicity.
In this case the iron binding is much worse when the amine functionality is diluted with hydroxyl functionality, a substitution that is not expected to make the polymer significantly more hydrophobic. This result suggests that dilution of the primary monomer is a factor and that hydrophobic/hydrophilic effects may be secondary. Dilution with acrylamide and phosphonic acid functionality also impacts negatively on the binding properties. In this case the negative charge expected on the phosphonic acid groups may inhibit binding of the negatively charged heme groups.
A variety of other amine-containing polymers was tested for heme-iron binding. The data on these polymers is shown below. Clearly the polyvinylamine is very effective (among the best), while the other polymers are less so. It is evident from these and other data that all types of amines (primary, secondary, tertiary, quaternary, and heterocyclic) can be made to bond heme-bound iron.
A variety of polymers with functional groups designed to bond directly to the iron atom within the heme were tested with the results shown below. Two of these, poly(AEABMP) and poly(AEABPHA), also contained an amine functionality that could be positively charged under the conditions of the iron-binding test. Thus they are capable of both direct binding and binding by ion exchange. Those polymers without this capability (the first six in the table below) were less effective than those two with it.
It might be expected that the extent of crosslinking could impact the heme-binding characteristics of these polymers. Since heme is a relatively large molecule it might have difficulty finding its way into a tightly crosslinked polymer gel. Alternatively, a too loosely crosslinked network might not effectively hold a heme molecule because of the potentially greater loss in entropy in binding to it. A highly crosslinked network might have cavities just large enough for a heme to fit tightly in, just as substrates fit in enzymatic active sites, while a less crosslinked (or even uncrosslinked) polymer may have to wrap itself around a heme with a significant loss in its internal entropy.
In order to partially assess such hypotheses two identical polymers with different amounts of crosslinking were synthesized. Poly(ammoniumbutylacrylamide) was synthesized with either 5% or 10% methylenebisacrylamide as crosslinked. The data below show that little difference was observed. Either there is little effect or extent of crosslinking on heme-iron binding, or the effects take place primarily outside of the range tested.
Two commercially available crosslinked polymeric materials that contain amine functionality are Questran(copyright) bile salt binder (cholestyramine; Bristol Laboratories) and Colestid(copyright) bile salt binder (colestipol; Upjohn). The structures of these polymers are shown below. 
Heme binding test results for these polymers are given in the following table. These products do demonstrate some heme-iron affinity, but they are not as effective as some of the polymers described above.
Heme iron binding was also tested for two of the polymers in the presence of a variety of potential small intestine contents. A test solution was made up with the following ingredients:
The pH was adjusted to 7.1 with acetic acid and some undissolved material was filtered off.
To this dark brown test solution was added 0.2 g of polymer. The solution was stirred 3 hours, during which time the pH shifted to xcx9c7.5 (and was not readjusted). The solid was filtered off and the iron content analyzed by atomic absorption spectroscopy at a commercial laboratory with the following results:
While it is evident that the polymers are not as effective as they are in the heme-iron only solution, they are still capable of binding a significant amount of heme.
Free Iron
One effective method of sequestering free iron involves attachment of classic iron chelators to a crosslinked polymer backbone. Iron chelators are typically small molecules that have between two and six subunits that attach themselves directly to the iron atom. Desferal(copyright) chelator (deferoxamine mesylate) is a good example. Good chelators contain such moieties as phenolates, enolic hydroxyls, ketones, aldehydes, carboxylates, phosphates and phosphonates, thiolates, sulfides and disulfides, hydroxamic acids and hydroxamates, amines, amides, and nitrones. The polymers can be designed such that the iron is chelated entirely by side chain groups 
or such that it is chelated at least partially across the backbone: 
In order to assess the potential of each candidate polymer a test was devised to quantitate the binding of iron to the polymer. This test involved stirring the polymer in a solution designed to mimic physiologic conditions. The amount of iron chosen corresponds to xcx9c9 mg of iron (a typical daily intake) and is dissolved in 1 L of fluid (the amount typically passing out of the small intestine in one day).
Results are shown below for a variety of polymers.
Clearly some of the polymers are more effective than others, with poly(vinylamine), poly(ethyleneimine), and poly(dimethylaminopropylmethacrylamide) being among the most effective.
Methods
Heme-Iron Assay
The polymer to be tested is ground and sieved to xe2x88x9280/+200 mesh size unless it is already a fine powder, in which case it is used as it. A measured amount of the polymer (typically 0.05-0.2 g) is suspended in 100 mL of the heme test solution. The pH is adjusted to 7.0 using either acetic acid or 1 N NaOH as necessary. The mixture is then stirred for three hours, at the end of which the pH is again adjusted to 7.0. The solid is then filtered off using Whatman #1 filter paper, and the liquid is examined spectroscopically.
Heme-bound iron has a broad absorption at xcx9c340-380 nm. The absorption is determined at 365 nm and corrected for a baseline absorption, typically by subtracting the average of the absorbances at 380 and 450 nm.
A635=A365(measured)xe2x88x92(A280+A450)/2 xe2x80x83xe2x80x83(1) 
The concentration of heme iron is then determined by comparison to a standard curve made using the starting solution and various dilutions thereof by plotting the relationship between corrected absorbance and the concentration of heme iron. The relationship generally fits well by a straight line of the formula:
[Heme Fe]=100%xc3x97[(0.189xc3x97A365)+0.001]xe2x80x83xe2x80x83(2) 
where the [Heme Fe] is the percent heme remaining by comparison to the starting heme solution.
Free Iron Assay
The free iron assay is similar to that used for heme iron. To 50 mL of the filtered iron test solution is added 3 mL to 0.3% aqueous o-phenanthroline and 1 mL of 10% aqueous hydroxylamine hydrochloride. The solution is stirred, and the pH is brought to 3.5 using aqueous sodium citrate (250 g/L) or 0.1N sulfuric acid, then diluted to a final volume of 60 mL. The solution is stirred for 5 minutes and then allowed to sit for 20 hours at room temperature. The absorbance is then read at 508 nm, with baseline points determined at 400 nm and 616 nm. The corrected absorbance at 508 nm is calculated by subtracting the average of the absorbances at 400 nm and 616 nm.
A508=A508(measured)xe2x88x92(A400+A616)/2 xe2x80x83xe2x80x83(3) 
The relationship between A408 and the free iron concentration is not a single straight line over the entire range of interest. The relationship is linear over three ranges and the linear least squares fits were used to derive the equations below:
where [Fe] is the % of free iron remaining compared to the original solution. Values of [Fe] below 2% are reported as xe2x80x9c less than 2xe2x80x9d % due to uncertainity in this range.